1. Field of the Invention
The present invention relates to a novel Paenibacillus sp. strain producing novel xylanase, a novel xylanase separated from the strain, and a method for mass-production of the same using the transformant of the strain. More precisely, the said novel xylanase exhibits excellent activity of decomposing xylan at high temperature or in a wide range of pH, so that the novel enzyme can be effectively used not only in the fields of feeds, paper and detergent industries, but also in the field of biochemical industry producing bio-fuel, petroleum alternative fuel, performance chemical, or bio-polymer.
2. Description of the Related Art
To keep pace with the changes of international environmental regulations including reduction of carbon dioxide emission, humankind of 21st century has the assignment to develop alternative resources of fossil fuel. In preparation of global warming and oil resource depletion, solar energy, wind power, hydropower, atomic power, and biomass have been major targets of research. In the process of biorefinery for the production of bio-fuel and chemical fuel via saccharification of lignocellulosic biomass, it is inevitable to produce the by-product, xylan, that is included in the lignocellulosic biomass by 15-30%. However, it is not possible to use the by-product directly, so most of it are discarded as wastes.
Hydrolysis of xylan has been induced via chemical method so far, which is precisely as follows: sulfuric acid is added to lignocellulosic biomass, followed by decomposing at 130° C. with pressurized steam; leading to the conversion into xylose and xyloologosaccharide. However, in the above process, many impurities are additionally generated by excessive reaction. Therefore, purifying technique is additionally requested. This method has other disadvantages as follows: it not only consumes a massive amount of energy but also requires high priced equipments durable in acid and high temperature condition, and it costs extra money for the treatment of wastes generated during the processes, raising the production cost as well. On the other hand, the biological method for xylan decomposition using xylanase (the enzyme that converts xylan, the major component of hemicellulose, into xylose via saccharification is generally called xylanase) consumes less energy and produces less wastes, compared with the chemical method, suggesting that it has an economical advantage owing to the easy treatment of less wastes.
Xylanase is not only urgently requested in the process of biorefinery (biochemical industry) but also utilized in the process of paper bleaching, for the improvement of feeds efficiency, in the clearing process of fruit beverages, for the production of high quality bread, and in the utilization of agricultural by-products, etc. Xylanase has been produced by using various microorganisms up to date. In particular, alkali-resistant or heat-resistant xylanase was isolated from various bacteria for the use in breaching process of paper industry (Tenkanen, M. et. al., Enzyme. Microb. Technol, 14, 566-574, 1992). Many xylanase producing microorganisms without cellulase activity have been reported, and attempts to reduce cellulose loss in the production of paper have been also reported (Khashin, A. et. al., Appl. Environ. Microbiol, 59, 1725-1730, 1993; Kosugi, A. et. al., J. Bacteriol, 183, 7037-7043, 2001). Successful cases have been reported to improve quality of bread by treating xylanase (Courtin, C. M. et. al., J. Agric. Food. Chem., 47, 1870-1877, 1999), and to introduce β-xylosidase and xylanase genes into yeast that is further used for saccharification of agricultural by-products for a microorganism to use them (La Grange, D. C. et. al., Appl. Environ. Microbiol, 67, 5512-5519, 2001). In the feeds industry, xylanase came into the market as a feed additive enzyme. So, when the cattle eat grain feeds containing the feed additive enzyme, the enzyme is functioning lower viscosity generated by hemicellulose of the intestines of the cattle, indicating that it is helpful to prevent digestive disease in the cattle and to improve feed efficiency (McCracken, K. J. et. al., Br. Poult. Sci, 42, 638-642, 2001).
Among the xylanase producing microorganisms reported so far, Trichoderma sp. fungal strains have been largely used whose enzyme productivity is superior than other xylanase producing bacteria but the maximum activity is mainly observed in acidic condition (Tenkanen at al., Enzyme and Microbial Technology 14(7):566-574, 1992). Xylanase producing bacteria are exemplified by Aeromonas sp., Bacillus sp., Clostridium sp., Streptomyces sp., Aspergillus sp, etc. The properties of xylanase depend on the bacteria and various genes able to encode xylanase have also been reported.
Status of domestic and international technology involved in xylanase is as follows. Trichoderma sp. C-4 strain producing cellulase was identified by Dr. Jung's research team of Kyung Hee University, Korea, however, higher activity is required for the industrialization (Sul et al., Appl Microbiol Biotechnol. 66(1):63-70, 2004). Cephalosporium sp. RYM-202 strain producing alkali-resistant xylanase was identified by Dr. Kang's research team of Donghae University, Korea, and its usability in pulp processing is being studied (Kang et al., Korean Journal of Environmental Biology 17(2):191-198, 1999). Bacillus subtilis DB104/pJHKJ4, the recombinant strain producing Bacillus originated endoxylanase, was constructed by Dr. Kim's research team of KAIST, Korea (Kim J H et al., J. Microbiol. Biotechnol., 10(4):551-553, 2000). In Taiwan, a case has been reported that alkali-resistance of xylanase genetically replicated from the anaerobic fungus Neocallimastix patriciarum was increased by directed enzyme evolution (Yew-Loom Chen et al., Can. J. Microbiol. 47(12):10881094, 2001) and recently Bacillus firmus, one of the alkali-resistant strains, has been identified in waste water generated from the pulp processing (Pochih Chang, Biochemical and Biophysical Research Communications 319:1017-1025, 2004). This xylanase demonstrates high activity in the wide pH range of 4˜11 and heat-resistance as high as maintaining 70% of the original activity even after 16 hour culture at 62° C. Likewise, various strains have been developed and their functions have been improved via directed evolution in many countries.
Patents in relation to xylanase so far are mainly focused on the method for producing xylanase by using a recombinant strain obtained from E. coli or using wild-type strain identified as xylanase producing one (International Patent Publication No. 93/08275, International Patent Publication No. 92/01793, International Patent Publication No. 92/17573, Korean Patent Publication No. 10-0072225, Korean Patent Publication No. 10-02211204, Korean Patent Publication No. 10-0411771). Patents in relation to xylanase as a feed additive so far are as follows: Novel Streptomyces sp. WL-2 strain producing xylanase (Korean Patent Publication No. 2001-0111986); Recombinant plasmid containing secretion signal sequence of endoxylanase from Bacillus subtilis and expression of foreign proteins using thereof (Korean Patent Publication No. 2000-0034279); and Gene coding xylanase of Bacillus sp. AMX-4 strain and recombinant xylanase through transformant thereof (Korean Patent Publication No. 2003-0085679).
Domestic patents in relation to xylanase so far are as follows: Novel Streptomyces sp. WL-2 strain producing xylanase (Korean Patent Publication No. 2001-0111986); Recombinant plasmid containing secretion signal sequence of endoxylanase from Bacillus subtilis and expression of foreign proteins using thereof (Korean Patent Publication No. 2000-0034279); Gene coding xylanase of Bacillus sp. AMX-4 strain and recombinant xylanase through transformant thereof (Korean Patent Publication No. 2003-0085679); and Novel Paenibacillus sp. HY-8 strain and xylanase isolated from it (Korean Patent Publication No. 2007-0082329). However, there was no specific explanation about the enzyme activity, neither was reported the cases of using them in domestic industry. Owing to the advanced technology, new methods to polymerize and produce various valuable compounds based on biomass such as high molecular compounds (plastic) have been developed and therefore it is urgently requested to develop xylanase with novel characteristics to match with the above.
For the efficient use of biomass, the development of saccharification process of cellulose using cellulase and the development of saccharification process of hemicellulose using xylanase need to be achieved at the same time. In the enzymatic saccharification process using xylanase, the characteristics of each enzyme required for the process are different according to the pre-treatment method of biomass. For example, in the process of pre-treatment of biomass using acid, acid-resistant saccharifying enzyme is required, while alkali-resistant saccharifying enzyme is required in the process of pre-treatment of biomass using alkali such as in the processes of pulp or paper production. In the meantime, heat-resistant enzyme is required for the simultaneous process of saccharification and fermentation. Most of commercialized xylanases are suitable for acid condition, indicating that novel xylanase demonstrating high activity under alkali condition needs to be developed. It is better to develop such xylanase that shows high activity in both acid and alkali conditions. To use xylanase directly in industry, it is important to secure xylanase gene that is able to maintain high activity under tough conditions such as high temperature and a wide range of pH via screening novel microorganisms and enzyme systems, from which the disadvantages shown in the conventional patents might be overcome.
To avoid those xylanases and those strains producing the same which have already been claimed by other advanced countries, not to infringe intellectual property right, the present inventors tried to develop novel xylanase to meet the domestic and further international need. In the course, the inventors completed this invention by confirming that the xylanase produced from the novel Paenibacillus sp. HPL-3 strain demonstrated excellent activity unit (Unit=mM product/mg protein/min), heat-resistance and a wide range of optimum pH, compared with the conventional xylanases, confirmed by solid culture and/or liquid culture enzyme activity measurement method.